Collagen is integral to, among other things, the proper formation of connective tissue. Therefore, the over- or under-production of collagen or the production of abnormal collagen (including incorrectly processed collagen) has been linked with numerous connective tissue diseases and disorders. Consequently, control and/or modulation of collagen formation has been the focus of study. These studies include efforts to identify enzymes, including C-proteinase, critical to collagen""s proper formation and processing.
The present invention is directed to the isolation and identification of the nucleic acid sequence encoding C-proteinase and the corresponding polypeptide, the recognition of such polypeptide activity, and applications, tools, processes and methods of use thereof.
Collagen Structure. At present, nineteen types of collagens have been identified. These collagens, including fibrillar collagen types I, II, III, are synthesized as procollagen precursor molecules which contain amino- and carboxy-terminal peptide extensions. These peptide extensions, referred to generally as xe2x80x9cpro-regions,xe2x80x9d are designated as N- and C-propeptides, respectively.
Both the N-propeptide and C-propeptide are typically cleaved upon secretion of the procollagen triple helical precursor molecule from the cell to yield a mature triple helical collagen molecule. Upon cleavage, the xe2x80x9cmaturexe2x80x9d collagen molecule is then capable of associating into highly structured collagen fibers. See e.g., Fessler and Fessler, 1978, Annu. Rev. Biochem. 47:129-162; Bornstein and Traub, 1979, in: The Proteins (eds. Neurath, H. and Hill, R. H.), Academic Press, New York, pp. 412-632; Kivirikko et al., 1984, in: Extracellular Matrix Biochemistry (eds. Piez, K. A. and Reddi, A. H.), Elsevier Science Publishing Co., Inc., New York, pp. 83-118; Prockop and Kivirikko, 1984, N. Engl. J. Med. 311:376-383; Kuhn, 1987, in: Structure and Function of Collagen Types (eds. Mayne, R. and Burgeson, R. E.), Academic Press, Inc., Orlando, Fla., pp. 1-42.
Diseases Associated With The Abnormal Production of Collagen. An array of critical diseases has been associated with the inappropriate or unregulated production of collagen, including pathological fibrosis or scarring, including endocardial sclerosis, idiopathic interstitial fibrosis, interstitial pulmonary fibrosis, perimuscular fibrosis, Symmers"" fibrosis, pericentral fibrosis, hepatitis, dermatofibroma, billary cirrhosis, alcoholic cirrhosis, acute pulmonary fibrosis, idiopathic pulmonary fibrosis, acute respiratory distress syndrome, kidney fibrosis/glomerulonephritis, kidney fibrosis/diabetic nephropathy, scleroderma/systemic, scleroderma/local, keloids, hypertrophic scars, severe joint adhesions/arthritis, myelofibrosis, corneal scarring, cystic fibrosis, muscular dystrophy (duchenne""s), cardiac fibrosis, muscular fibrosis/retinal separation, esophageal stricture, payronles disease. Further fibrotic disorders may be induced or initiated by surgery, including scar revision/plastic surgeries, glaucoma, cataract fibrosis, corneal scarring, joint adhesions, graft vs. host disease, tendon surgery, nerve entrapment, dupuytren""s contracture, OB/GYN adhesions/fibrosis, pelvic adhesions, peridural fibrosis, restenosis.
One strategy for the treatment of these diseases is the inhibition of the pathological overproduction of collagen. The identification and isolation of enzymes involved in the collagen production and processing are therefore of major medical interest to provide for suitable targets for drug development.
Similarly, a strategy for the treatment of diseases resulting from the pathological underproduction of collagen, where the underproduction of collagen is the consequence of improper processing of procollagen, is the administration of C-proteinase.
Background Information Regarding C-Proteinase. C-proteinase is an enzyme that catalyzes the cleavage of the C-propeptide of fibrillar collagens, including type I, type II, and type III collagen. The enzyme was first observed in culture media of human and mouse fibroblasts (Goldberg et al., 1975, Cell 4:45-50; Kessler and Goldberg, 1978, Anal. Biochem. 86:463-469), and chick tendon fibroblasts (Duskin et al., 1978, Arch. Biochem. Biophys. 185:326-332; Leung et al., 1979, J. Biol. Chem. 254:224-232). An acidic proteinase which removes the C-terminal propeptides from type I procollagen has also been identified. Davidson et al., 1979, Eur. J. Biochem. 100:551.
A partially purified protein having C-proteinase activity was obtained from chick calvaria in 1982. Njieha et al., 1982, Biochemistry 23:757-764. In 1985, natural C-proteinase was isolated, purified and characterized from conditioned media of chick embryo tendons. Hojima et al., 1985, J. Biol. Chem. 260:15996-16003. Murine C-proteinase has been subsequently purified from media of cultured mouse fibroblasts. Kessler et al., 1986, Collagen Relat. Res. 6:249-266; Kessler and Adar, 1989, Eur. J. Biochem. 186:115-121.
Experiments conducted with these purified forms of chick and mouse C-proteinase have indicated that the enzyme is instrumental in the formation of functional collagen fibers. Fertala et al., 1994, J. Biol. Chem. 269:11584.
Generally, C-proteinase activity and the inhibition of the enzyme""s activity have been determined using a wide array of assays. See e.g., Kessler and Goldberg, 1978, Anal. Biochem. 86:463; Njieha et al., 1982, Biochemistry 21:757-764. As articulated in numerous publications, the enzyme is difficult to isolate by conventional biochemical means and neither the enzyme nor the cDNA sequence encoding such enzyme was known to be available prior to the instant invention. Takahara et al., 1994, J. Biol. Chem. 269:26280-26285, 26284 (C-proteinase""s xe2x80x9cpeptide and nucleotide sequences are as yet unavailablexe2x80x9d). Thus, despite the availability of C-proteinase related assays, large scale review and testing of potential C-proteinase inhibitors has not been performed to date.
Known C-Proteinase Inhibitors. A number of potential C-proteinase inhibitors have been identified. For example, several metal chelators have demonstrated activity as a C-proteinase inhibitor. Likewise, chymostatin and pepstatin A have been found to act as relatively strong inhibitors of C-proteinase activity.
xcex12-Macroglobulin, ovostatin, and fetal bovine serum appear to also, at least partially, inhibit C-proteinase activity. Similarly, dithiothreitol, SDS, concanavalin A, Zn2+, Cu2+, and Cd2+possess inhibitory activity at low concentrations, and some reducing agents, several amino acids (including lysine and arginine), phosphate, and ammonium sulfate have been found to have C-proteinase inhibitory activity at concentrations of 1-10 mM. Leung et al., supra; Ryhxc3xa4nen et al., 1982, Arch. Biochem. Biophys. 215:230-236.
High concentrations of NaCl or Tris-HCl buffer have also been found to inhibit the C-proteinase activity. For example, it has been reported that 0.2, 0.3, and 0.5M NaCl reduces the activity of C-proteinase by 66, 38, and 25%, respectively, of that observed with the standard assay concentration of 0.15M. Tris-HCl buffer in a concentration of 0.2-0.5M likewise has been reported to inhibit the enzyme""s activity. Hojima et al., supra.
In contrast, microbial inhibitors such as leupeptin, phosphoramidon, antipain, bestatin, elastinal, and amastatin, are considered to have weak or no effect.
Background Information Regarding Bone Morphogenic Protein-1 (BMP-1). A protein having the structural characteristics of C-proteinase was isolated in 1988 from bone tissue. Prior to the instant invention, it was believed that this protein, designated BMP-1 or xe2x80x9cbone morphogenic protein,xe2x80x9d was a member of the TGF-xcex2 related protein family (Wozney et al., 1988, Science 242:1528-1534), as BMP-1 was isolated coincidentally with BMP-2A and BMP-3. Although evidence provides that BMP-2A and BMP-3 play a key role in the stimulation of bone development and growth, the activity of BMP-1 was never clearly established.
Sequence comparison reveals that BMP-1 contains a EGF-like domain and a region designated as xe2x80x9cA-domainxe2x80x9d having sequence similarity with a protease isolated from crayfish. Titany et al., 1987, Biochemistry 26:222. As the TGF-xcex21 binding protein also contains EGF-like domains, it has been suggested that BMP-1 could be a protease involved in the activation of TGF-xcex21. Miyazono et al., 1988, J. Biol. Chem. 263:6407; Woyznek et al., supra; Fukagawa et al., 1994, Dev. Bio. 162:175-183.
It has also been suggested that, due to homology to the Drosophila melanogaster tolloid gene product, BMP-1 is involved in the overall mechanism for the dorsal-ventral patterning of the neural tube.
While it has been suggested that C-proteinase (xe2x80x9cfor which [prior to this invention] peptide and nucleotide sequence are as yet unavailablexe2x80x9d) and BMP-1 belong to the same structural family, BMP-1 has never been associated with the formation of collagen. Takahara et al., 1994, J. Biol. Chem. 269:26280-26286. Thus, while a cDNA and polypeptide sequence of the putative bone morphogenic protein BMP-1 had been identified, no correct activity or use was known for this protein until the present invention. Similarly, the structural relationship between BMP-1 and C-proteinase was not known.
The present invention is directed to synthesized or recombinant compositions derived from the deduced amino acid and nucleic acid sequences for human C-proteinase. In one embodiment of the present invention, the composition comprises the full-length amino acid sequence for C-proteinase. In another embodiment of the present invention, the composition comprises a C-proteinase derivative having C-proteinase-like activity. In yet further embodiments of the present invention, the composition is radiolabeled or represents an analog of C-proteinase having C-proteinase-like activity. The present invention is also related to the recombinant production of C-proteinase and related compositions in a variety of recombinant expression systems.
The present invention also relates to the use C-proteinase, its fragments, analogs and derivatives for use in diseases and disorders related to the abnormal production of collagen. Such polypeptides may act directly with collagen, or alternatively with other enzymes involved in the processing of collagen, i.e., lysyl oxidase.
The present invention also relates to the use of proteins, peptides and organic molecules capable of modulating the formation of collagen by affecting the interaction between C-proteinase and collagen precursor molecules, including procollagen, or alternatively, other collagen processing enzymes and/or the cleavage site of C-proteinase. The invention is further directed to the use of such proteins, peptides and/or organic molecules, either alone or in combination with other molecules, in the treatment of disorders, including disorders related to abnormal collagen formation, such as rheumatoid arthritis and scleroderma, for example.
The present invention is also related to the use of C-proteinase, whether labeled or unlabeled, as a tracer which could then be used to separate, by HPLC, the different C-proteinase derivatives to yield a carrier-free tracer, in binding assays.
Finally, the present invention is related to the recombinant expression and production of C-proteinase by use of the sequences of the invention.
xe2x80x9cC-proteinasexe2x80x9d shall be construed to mean an enzyme capable of processing collagen molecules, derivatives or fragments, or their precursors by cleaving through -Alas↓Asp-Asp- and/or -Gly↓Asp-Glu-. The term shall include human C-proteinase and derivatives, analogs, fragments and variants thereof having C-proteinase-like activity.